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- EMDB-0246: Leucine-zippered human insulin receptor ectodomain with single bo... -

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Basic information

Entry
Database: EMDB / ID: EMD-0246
TitleLeucine-zippered human insulin receptor ectodomain with single bound insulin - "lower" membrane-proximal part
Map datainsulin bound to insulin receptor ectodomain - "lower" membrane-proximal part
Sample
  • Complex: Leucine zippered human insulin receptor ectodomain (IR-A isoform, "deltabeta" mutant) in complex with insulin and two Fv 83-7 modules : "lower" membrane-proximal part
    • Protein or peptide: Insulin receptor,Insulin receptor,General control protein GCN4
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose
Keywordsinsulin / insulin receptor ectodomain / signal transdution / SIGNALING PROTEIN
Function / homology
Function and homology information


FCERI mediated MAPK activation / protein localization to nuclear periphery / regulation of female gonad development / Activation of the AP-1 family of transcription factors / positive regulation of meiotic cell cycle / response to amino acid starvation / negative regulation of ribosomal protein gene transcription by RNA polymerase II / positive regulation of cellular response to amino acid starvation / mediator complex binding / insulin-like growth factor II binding ...FCERI mediated MAPK activation / protein localization to nuclear periphery / regulation of female gonad development / Activation of the AP-1 family of transcription factors / positive regulation of meiotic cell cycle / response to amino acid starvation / negative regulation of ribosomal protein gene transcription by RNA polymerase II / positive regulation of cellular response to amino acid starvation / mediator complex binding / insulin-like growth factor II binding / positive regulation of developmental growth / male sex determination / insulin receptor complex / insulin-like growth factor I binding / positive regulation of protein-containing complex disassembly / insulin receptor activity / Oxidative Stress Induced Senescence / exocrine pancreas development / dendritic spine maintenance / cargo receptor activity / insulin binding / adrenal gland development / neuronal cell body membrane / PTB domain binding / Signaling by Insulin receptor / IRS activation / positive regulation of respiratory burst / amyloid-beta clearance / TFIID-class transcription factor complex binding / amino acid biosynthetic process / positive regulation of receptor internalization / regulation of embryonic development / insulin receptor substrate binding / positive regulation of RNA polymerase II transcription preinitiation complex assembly / protein kinase activator activity / epidermis development / positive regulation of glycogen biosynthetic process / positive regulation of transcription initiation by RNA polymerase II / Signal attenuation / heart morphogenesis / cellular response to nutrient levels / transport across blood-brain barrier / phosphatidylinositol 3-kinase binding / Insulin receptor recycling / insulin-like growth factor receptor binding / dendrite membrane / neuron projection maintenance / positive regulation of mitotic nuclear division / Insulin receptor signalling cascade / receptor-mediated endocytosis / cellular response to amino acid starvation / positive regulation of glycolytic process / positive regulation of D-glucose import / learning / receptor protein-tyrosine kinase / caveola / cellular response to growth factor stimulus / receptor internalization / RNA polymerase II transcription regulator complex / memory / male gonad development / cellular response to insulin stimulus / positive regulation of nitric oxide biosynthetic process / insulin receptor signaling pathway / late endosome / glucose homeostasis / amyloid-beta binding / protein autophosphorylation / PI5P, PP2A and IER3 Regulate PI3K/AKT Signaling / DNA-binding transcription activator activity, RNA polymerase II-specific / protein tyrosine kinase activity / transcription regulator complex / sequence-specific DNA binding / RNA polymerase II-specific DNA-binding transcription factor binding / DNA-binding transcription factor activity, RNA polymerase II-specific / lysosome / positive regulation of canonical NF-kappaB signal transduction / positive regulation of phosphatidylinositol 3-kinase/protein kinase B signal transduction / receptor complex / endosome membrane / positive regulation of MAPK cascade / intracellular signal transduction / positive regulation of cell migration / RNA polymerase II cis-regulatory region sequence-specific DNA binding / G protein-coupled receptor signaling pathway / DNA-binding transcription factor activity / protein domain specific binding / axon / external side of plasma membrane / positive regulation of cell population proliferation / chromatin binding / regulation of DNA-templated transcription / symbiont entry into host cell / GTP binding / protein-containing complex binding / positive regulation of DNA-templated transcription / negative regulation of transcription by RNA polymerase II / positive regulation of transcription by RNA polymerase II / extracellular exosome / ATP binding
Similarity search - Function
Insulin receptor, trans-membrane domain / Insulin receptor trans-membrane segment / : / Basic region leucine zipper / Tyrosine-protein kinase, insulin-like receptor / Basic-leucine zipper (bZIP) domain signature. / Basic-leucine zipper (bZIP) domain profile. / basic region leucin zipper / Basic-leucine zipper domain / Basic-leucine zipper domain superfamily ...Insulin receptor, trans-membrane domain / Insulin receptor trans-membrane segment / : / Basic region leucine zipper / Tyrosine-protein kinase, insulin-like receptor / Basic-leucine zipper (bZIP) domain signature. / Basic-leucine zipper (bZIP) domain profile. / basic region leucin zipper / Basic-leucine zipper domain / Basic-leucine zipper domain superfamily / Tyrosine-protein kinase, receptor class II, conserved site / Receptor tyrosine kinase class II signature. / Receptor L-domain / Furin-like cysteine-rich domain / Receptor L-domain superfamily / Furin-like cysteine rich region / Receptor L domain / Furin-like repeat / Furin-like repeats / Fibronectin type III domain / Growth factor receptor cysteine-rich domain superfamily / : / Fibronectin type 3 domain / Fibronectin type-III domain profile. / Fibronectin type III / Fibronectin type III superfamily / Tyrosine-protein kinase, catalytic domain / Tyrosine kinase, catalytic domain / Tyrosine protein kinases specific active-site signature. / Tyrosine-protein kinase, active site / Serine-threonine/tyrosine-protein kinase, catalytic domain / Protein tyrosine and serine/threonine kinase / Protein kinase, ATP binding site / Protein kinases ATP-binding region signature. / Immunoglobulin-like fold / Protein kinase domain profile. / Protein kinase domain / Protein kinase-like domain superfamily
Similarity search - Domain/homology
General control transcription factor GCN4 / Insulin receptor
Similarity search - Component
Biological speciesHomo sapiens (human) / Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.2 Å
AuthorsWeis F / Menting JG
Funding support Australia, United States, 4 items
OrganizationGrant numberCountry
National Health and Medical Research Council (Australia)APP1099595 Australia
National Health and Medical Research Council (Australia)APP1128553 Australia
National Institutes of Health/National Institute of Diabetes and Digestive and Kidney Disease (NIH/NIDDK)DK013914 United States
National Institutes of Health/National Institute of Diabetes and Digestive and Kidney Disease (NIH/NIDDK)UC DRTC DK020595 United States
CitationJournal: Nat Commun / Year: 2018
Title: The signalling conformation of the insulin receptor ectodomain.
Authors: Felix Weis / John G Menting / Mai B Margetts / Shu Jin Chan / Yibin Xu / Norbert Tennagels / Paulus Wohlfart / Thomas Langer / Christoph W Müller / Matthias K Dreyer / Michael C Lawrence /
Abstract: Understanding the structural biology of the insulin receptor and how it signals is of key importance in the development of insulin analogs to treat diabetes. We report here a cryo-electron microscopy ...Understanding the structural biology of the insulin receptor and how it signals is of key importance in the development of insulin analogs to treat diabetes. We report here a cryo-electron microscopy structure of a single insulin bound to a physiologically relevant, high-affinity version of the receptor ectodomain, the latter generated through attachment of C-terminal leucine zipper elements to overcome the conformational flexibility associated with ectodomain truncation. The resolution of the cryo-electron microscopy maps is 3.2 Å in the insulin-binding region and 4.2 Å in the membrane-proximal region. The structure reveals how the membrane proximal domains of the receptor come together to effect signalling and how insulin's negative cooperativity of binding likely arises. Our structure further provides insight into the high affinity of certain super-mitogenic insulins. Together, these findings provide a new platform for insulin analog investigation and design.
History
DepositionSep 14, 2018-
Header (metadata) releaseNov 21, 2018-
Map releaseNov 21, 2018-
UpdateNov 13, 2024-
Current statusNov 13, 2024Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.03
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.03
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  • Surface view with fitted model
  • Atomic models: PDB-6hn4
  • Surface level: 0.03
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_0246.png

Downloads & links

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Map

FileDownload / File: emd_0246.map.gz / Format: CCP4 / Size: 91.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationinsulin bound to insulin receptor ectodomain - "lower" membrane-proximal part
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.04 Å/pix.
x 288 pix.
= 299.52 Å		1.04 Å/pix.
x 288 pix.
= 299.52 Å		1.04 Å/pix.
x 288 pix.
= 299.52 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.04 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.03 / Movie #1: 0.03
Minimum - Maximum-0.054737736 - 0.10128318
Average (Standard dev.)0.00016646631 (±0.002360165)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions288288288
Spacing288288288
CellA=B=C: 299.52 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.041.041.04
M x/y/z288288288
origin x/y/z0.0000.0000.000
length x/y/z299.520299.520299.520
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ240240240
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS288288288
D min/max/mean-0.0550.1010.000

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Supplemental data

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Sample components

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Entire : Leucine zippered human insulin receptor ectodomain (IR-A isoform,...

EntireName: Leucine zippered human insulin receptor ectodomain (IR-A isoform, "deltabeta" mutant) in complex with insulin and two Fv 83-7 modules : "lower" membrane-proximal part
Components
  • Complex: Leucine zippered human insulin receptor ectodomain (IR-A isoform, "deltabeta" mutant) in complex with insulin and two Fv 83-7 modules : "lower" membrane-proximal part
    • Protein or peptide: Insulin receptor,Insulin receptor,General control protein GCN4
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose

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Supramolecule #1: Leucine zippered human insulin receptor ectodomain (IR-A isoform,...

SupramoleculeName: Leucine zippered human insulin receptor ectodomain (IR-A isoform, "deltabeta" mutant) in complex with insulin and two Fv 83-7 modules : "lower" membrane-proximal part
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Details: Note: Attached to the leucine-zippered insulin receptor ectodomain are two Fv 83-7 modules. One of these is present within this map volume but it is very poorly ordered and thus left ...Details: Note: Attached to the leucine-zippered insulin receptor ectodomain are two Fv 83-7 modules. One of these is present within this map volume but it is very poorly ordered and thus left completely unmodelled. See the manuscript for further details.
Source (natural)Organism: Homo sapiens (human)

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Macromolecule #1: Insulin receptor,Insulin receptor,General control protein GCN4

MacromoleculeName: Insulin receptor,Insulin receptor,General control protein GCN4
type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO / EC number: receptor protein-tyrosine kinase
Source (natural)Organism: Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Strain: ATCC 204508 / S288c
Molecular weightTheoretical: 106.728211 KDa
Recombinant expressionOrganism: Cricetulus griseus (Chinese hamster)
SequenceString: HLYPGEVCPG MDIRNNLTRL HELENCSVIE GHLQILLMFK TRPEDFRDLS FPKLIMITDY LLLFRVYGLE SLKDLFPNLT VIRGSRLFF NYALVIFEMV HLKELGLYNL MNITRGSVRI EKNNELCYLA TIDWSRILDS VEDNHIVLNK DDNEECGDIC P GTAKGKTN ...String:
HLYPGEVCPG MDIRNNLTRL HELENCSVIE GHLQILLMFK TRPEDFRDLS FPKLIMITDY LLLFRVYGLE SLKDLFPNLT VIRGSRLFF NYALVIFEMV HLKELGLYNL MNITRGSVRI EKNNELCYLA TIDWSRILDS VEDNHIVLNK DDNEECGDIC P GTAKGKTN CPATVINGQF VERCWTHSHC QKVCPTICKS HGCTAEGLCC HSECLGNCSQ PDDPTKCVAC RNFYLDGRCV ET CPPPYYH FQDWRCVNFS FCQDLHHKCK NSRRQGCHQY VIHNNKCIPE CPSGYTMNSS NLLCTPCLGP CPKVCHLLEG EKT IDSVTS AQELRGCTVI NGSLIINIRG GNNLAAELEA NLGLIEEISG YLKIRRSYAL VSLSFFRKLR LIRGETLEIG NYSF YALDN QNLRQLWDWS KHNLTTTQGK LFFHYNPKLC LSEIHKMEEV SGTKGRQERN DIALKTNGDK ASCENELLKF SYIRT SFDK ILLRWEPYWP PDFRDLLGFM LFYKEAPYQN VTEFDGQDAC GSNSWTVVDI DPPLRSNDPK SQNHPGWLMR GLKPWT QYA IFVKTLVTFS DERRTYGAKS DIIYVQTDAT NPSVPLDPIS VSNSSSQIIL KWKPPSDPNG NITHYLVFWE RQAEDSE LF ELDYCLKGLK LPSRTWSPPF ESEDSQKHNQ SEYEDSAGEC CSCPKTDSQI LKELEESSFR KTFEDYLHNV VFVPRPSR K RRSLGDVGNA GNNEEHRPFE KVVNKESLVI SGLRHFTGYR IELQACNQDT PEERCSVAAY VSARTMPEAK ADDIVGPVT HEIFENNVVH LMWQEPKEPN GLIVLYEVSY RRYGDEELHL CVSRKHFALE RGCRLRGLSP GNYSVRIRAT SLAGNGSWTE PTYFYVTDY LDVPSNIARM KQLEDKVEEL LSKNYHLENE VARLKKLVGE R

UniProtKB: Insulin receptor, Insulin receptor, General control transcription factor GCN4

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Macromolecule #2: 2-acetamido-2-deoxy-beta-D-glucopyranose

MacromoleculeName: 2-acetamido-2-deoxy-beta-D-glucopyranose / type: ligand / ID: 2 / Number of copies: 6 / Formula: NAG
Molecular weightTheoretical: 221.208 Da
Chemical component information

ChemComp-NAG:
2-acetamido-2-deoxy-beta-D-glucopyranose

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration0.094 mg/mL
BufferpH: 7.5
Component:
ConcentrationFormulaName
10.0 mMHEPES-NaOH
0.02 %sodium azide
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 283.15 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Specialist opticsEnergy filter - Name: GIF Quantum LS / Energy filter - Slit width: 20 eV
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: SUPER-RESOLUTION / Digitization - Frames/image: 1-20 / Number grids imaged: 1 / Number real images: 2287 / Average exposure time: 16.0 sec. / Average electron dose: 1.85 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.5 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 130000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 747074
Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

4zxb


Details: symmetrised domains L1, CR and L2 bound to Fv 83-7
Final reconstructionNumber classes used: 2 / Applied symmetry - Point group: C1 (asymmetric) / Algorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 4.2 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 2.1) / Number images used: 98481
Initial angle assignmentType: PROJECTION MATCHING / Software - Name: RELION (ver. 2.1)
Final angle assignmentType: PROJECTION MATCHING / Software - Name: RELION (ver. 2.1)
Final 3D classificationNumber classes: 6 / Software - Name: RELION (ver. 2.1)
Details: performed with partial signal subtraction of the "upper" part.

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Atomic model buiding 1

Initial modelPDB ID:

4zxb


Chain - Source name: PDB / Chain - Initial model type: experimental model
RefinementSpace: REAL / Protocol: OTHER / Target criteria: Cross-correlation coefficient
Output model

PDB-6hn4:
Leucine-zippered human insulin receptor ectodomain with single bound insulin - "lower" membrane-proximal part

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